1. Field of the Invention
The present invention is directed to a method of inhibiting gene expression promoted by the AP1 protein complex, with compounds which specifically or selectively transactivate RARxcex2 retinoid receptors. The present invention is also directed to a method of administering pharmaceutical compositions for the treatment or prevention of certain diseases and conditions which comprise a compound or ligand capable of inhibiting gene expression promoted by the AP1 protein complex through RARs, and which compound specifically or selectively induces gene expression only through the RARxcex2 retinoid receptors. The present invention is further directed to a method of selecting compounds of beneficial retinoid-like activity by assaying candidate compounds for ability to antagonize the gene expression medicated through the AP1 protein complex to suppress the expression of gene promoted by the AP1 protein complex, and for the ability, or lack thereof of the compounds to transactivate gene expression through RARxcex1, RARxcex2 and RARxcex93 receptors.
2. Related Art
Compounds having retinoic acid like (retinoid-like) biological activity have been known for a long time, and are described in numerous United States and foreign patents and scientific publications. Generally speaking, it has been established and accepted in the art that retinoic-acid like compounds (retinoids) are useful in humans and domestic animals for the treatment or prevention of many diseases and conditions, as regulators of cell proliferation and differentiation, and particularly as agents for treating dermatoses, such as acne, Darier""s disease, psoriasis, icthyosis, eczema and atopic dermatitis, and for treating and preventing malignant hyperproliferative diseases such as epithelial cancer, breast cancer, prostatic cancer, head and neck cancer and myeloid leukemias, for reversing and preventing atherosclerosis and restenosis resulting from neointimal hyperproliferation, for treating and preventing other non-malignant hyperproliferative diseases such as endometrial hyperplasia, benign prostatic hypertrophy, proliferative vitreal retinopathy and dysplasias, for treating autoimmune diseases and immunological disorders (e.g. lupus erythematosus) for treating chronic inflammatory diseases such as pulmonary fibrosis, for treating and preventing diseases associated with lipid metabolism and transport such as dyslipidemias, for promoting wound healing, for treating dry eye syndrome and for reversing and preventing the effects of sun damage to skin.
A classic measure of retinoic-acid like activity involves the effects of retinoic acid on ornithine decarboxylase. The original work on the correlation between retinoic acid and decrease in cell proliferation was done by Verma and Boutwell, Cancer Research, 1977, 37,2196-2201. That reference discloses that ornithine decarboxylase (ODC) activity increased precedent to polyamine biosynthesis. It has been established elsewhere that increases in polyamine synthesis can be correlated or associated with cellular proliferation. Thus, it was recognized early in the art that if ODC activity could be inhibited, cell hyperproliferation could be modulated. Although all causes for ODC activity increases are unknown, it is known that 12-0-tetradecanoylphorbol-13-acetate (TPA) induces ODC activity. Retinoic acid inhibits this induction of ODC activity by TPA. An assay essentially following the procedure set out in Cancer Res: 1662-1670,1975 has been extensively used in the art to demonstrate inhibition of TPA induction of ODC by tests compounds, which compound, if found inhibitory in the assay, is considered a xe2x80x9cretinoidxe2x80x9d. Several other assays also exist in the art to determine if a compound has retinoid-like biological activity.
The use of retinoids in therapy of humans or domestic animals, is however, usually not without unpleasant or even serious side effects. Therefore, efforts have been made in the art to develop compounds which retain the beneficial activity of retinoids, but nevertheless lack the undesired side effects. U.S. Pat. No. 5,324,840 (assigned to the same assignee as the present application) for example discloses compounds which have retinoid-like activity but either lack, or have diminished skin-toxicity or teratogenic activity. An application for United States patent titled xe2x80x9cMethod of Treatment with Compounds Having Selective Agonist-like Activity on RXR Retinoid Receptorsxe2x80x9d Ser. No. 08/016,404 has been allowed and is expected to issue.
Significant efforts have also been made in the prior art to elucidate, on a biologiocal, pharmacological or molecular level, the mechanisms by which retinoids act in living organisms, and specifically by which retinoids act to bring about beneficial therapeutic results and the undesired side effects. In connection with the foregoing it has been established in the prior art that one mode of action of retinoids is inducing gene expression through a class of receptors which are termed xe2x80x9cRARxe2x80x9d, another is through a class of receptors termed xe2x80x9cRXRxe2x80x9d. Both the RAR and RXR receptor xe2x80x9cfamiliesxe2x80x9d have been shown to include several subtypes, which in the case of the RAR receptors are termed RARxcex1, RARxcex2 and RARxcex93. Generally speaking, the following publications pertain to retinoid receptors and/or to compounds for selectively activating one or more of the receptor subtypes: D. J. Mangelsdorf et al. xe2x80x9cNuclear receptor that identifies a novel retinoic acid response pathwayxe2x80x9d, Nature Vol 345 May 17, 1990 pp 224-229; and J. N. Rottman et al. A retinoic acid-responsive element in the apiloprotein AI gene distinguishes between two different retinoic acid response pathways, Molecular and Cellular Biology, July 1991, pp 3814-3820, M. Petkovich et al. xe2x80x9cA human retinoic acid receptor which belongs to the family of nuclear receptorxe2x80x9d, Nature, Vol. 330, Dec. 3, 1987, pp 444-450; V. Giguere et al. xe2x80x9cIdentification of a receptor for the morphogen retinoic acidxe2x80x9d, Nature, Vol 330, Dec. 17, 1987, pp 624-629; N. Brand et al. xe2x80x9cIdentification of a second human retinoic acid receptorxe2x80x9d, Nature, Vol 332, Apr. 28, 1988, pp 850-853; A. Krast et al., xe2x80x9cA third human retinoic acid receptor, hRARxe2x80x9d, Proc. Natl. Acad. Sci. USA, Vol 86, July 1989, pp 5310-5314; D. J. Mangelsdorf et al., xe2x80x9cCharacterization of three RXR genes that mediate the action of 9-cis-retinoic acidxe2x80x9d, Genes and Development, Vol. 6, 1992, pp. 329-344, D. J. Mangelsdorf et al. xe2x80x9cNuclear receptor that identifies a novel retinoic acid response pathwayxe2x80x9d Nature Vol. 345, May 17 1990, pp224-229, and International Publication WO 93 21146 (Ligand Pharmaceuticals) titled xe2x80x9cCompounds Having Selectivity for Retinoid X Receptorsxe2x80x9d.
As a still further development in the art pertaining to the mechanism of action of retinoids at the pharmacological and molecular level, it has been discovered that in the presence of a proper ligand (retinoid compound), the RARs regulate gene expression either by directly binding to the RA-responsive element (RARE) or by antagonizing the action of c-Jun/c-Fos (AP1) protein complex. More specifically, in the action of a retinoid (ligand) that acts through the RARs, one or more of the three different RAR subtypes (RARxcex1, RARxcex2 and RARxcex93) bind to the retinoid ligand. The resulting RAR-ligand complex regulates gene expression either by activating the expression of genes containing RAREs in their promoter regions, or by inhibiting the expression of certain genes by antagonizing AP1 protein complex (c-Jun/c-Fos, hereinafter xe2x80x9cAP1 proteinxe2x80x9d or xe2x80x9cAP1 protein complexxe2x80x9d) mediated gene expression. (See the articles: Mangelsdorf et al. (1994). The Retinoids; Biology, Chemistry, and Medicine, pp. 219-349. Raven Press Ltd., New York; Chambon, P. (1994) Semin, Cell Biol. 5, 115-125, and Pfahl. M. (1993) Endocr. Rev. 14, 651-658). Antagonizing the AP1 protein and thereby suppressing or inhibiting expression of the AP1-promoted gene is considered a second mechanism of action of retinoids. The latter is generally considered to be beneficial from a therapeutic standpoint, because the genes stimulated by the AP1 protein are involved in hyperproliferative and inflammatory diseases such as psoriasis, rheumatoid arthritis and tumor metastases. Published International Patent Application Nos. WO 92/05447 and WO92/07072 relate to the subject of inhibiting the gene expression which is stimulated by the AP1 protein complex.
In contrast to the action of retinoid compounds (ligands) to prevent or inhibit expression of the gene promoted by AP1, the transactivation of genes containing the RAREs, on the other hand, can lead to some of the undesired side-effect of retinoids. Therefore, a need exists in the prior art to separate the two types of retinoid actions, whereby retinoid drugs of lesser toxicity and therefore of greater therapeutic benefit may be obtained.
The present invention comprises a method for binding AP1 protein in a complex with a retinoid receptor RAR which has been activated by a retinoid compound or ligand, thereby inhibiting expression of the gene promoted by the AP1 protein, in preference over activating expression of genes which include the RAREs in their promoter regions. In other words, the present invention comprises a method for selectively inhibiting expression of the gene which is promoted by the AP1 protein, without practically inducing expression of genes that are normally triggered by retinoid like compounds through the RAREs.
In another aspect, the present invention comprises the above-mentioned gene regulation method employed for therapeutic purposes with reduced side effects, such as regulation of cell proliferation and differentiation, and particularly for treating dermatoses, such as acne, Darier""s disease, psoriasis, icthyosis, eczema, atopic dermatitis, and for treating and preventing malignant hyperproliferative diseases such as epithelial cancer, breast cancer, prostatic cancer, head and neck cancer and myeloid leukemias, for reversing and preventing artherosclerosis and restenosis resulting from neointimal hyperproliferation, for treating and preventing other non-malignant hyperproliferative diseases such as endometrial hyperplasia, benign prostatic hypertrophy, proliferative vitreal retinopathy and dysplasias, for treating autoimmune diseases and immunological disorders (e.g. lupus erythematosus), for treating arthritis, asthma, allergies, chronic inflammatory diseases such as pulmonary fibrosis, for treating and preventing diseases associated with lipid metabolism and transport such as dyslipidemias, for promoting wound healing, for treating dry eye syndrome and in reversing and preventing the effects of sun damage to skin.
In still another aspect, the present invention comprises pharmaceutical compositions with which the above-noted methods are practiced, and which contain retinoid-like compounds capable of inhibiting AP1-promoted gene expression without causing expression of practical significance of genes having RAREs in their promoter region.
In yet another aspect, the present invention comprises a method for selecting retinoid-compounds as candidates for drugs having reduced side effects, by assaying a candidate test compound for its ability to repress AP1-protein promoted gene expression through the three RAR subtypes (RARxcex1, RARxcex2 and RARxcex93) and for assaying the candidate test compound for its ability to transactivate gene expression through one or more of the three RARs. Compounds which inhibit AP1-protein expression through all three RAR subtypes, but transactivate only through the RARxcex2 receptor are selected, because the RARxcex2 receptor is practically not present in mammalian skin, and therefore side effects in the skin by the use of such compound are practically avoided.
In a further aspect, the present invention relates to the method of using the compounds which have the foregoing properties for cancer chemotherapy.